Asthma is a leading cause of morbidity and mortality affecting more that 300 million people worldwide. While numerous therapies exist to treat airway inflammation and bronchospasm symptomatically, gene therapy can alter the molecular dysfunctions driving disease pathogenesis. Unfortunately, this therapeutic approach has been hindered by the inability to selectively transfer genes to non-dividing vascular and airway smooth muscle cells without inducing damage. Our ongoing goal is to design more effective gene therapy vectors by elucidating the molecular mechanisms that transport DNA and DNA-protein complexes actively and selectively into the nucleus of specific cell types. We have identified a DNA sequence derived from the smooth muscle gamma actin (SMGA) promoter that increases nuclear localization and subsequent gene expression uniquely in smooth muscle cells, a critical target in asthma gene therapy. We have shown that the cell-selective nuclear import of the SMGA DNA nuclear targeting sequence (or DTS) is mediated by the transcription factors SRF and Nkx3.1/3.2 that are uniquely co-expressed in smooth muscle cells but not other cells of the airway. In recent proteomics-based studies we identified a number of candidate proteins that may be involved in trafficking of the DNA protein complexes in smooth muscle cells. Further, we have developed a new technique for in vivo gene delivery using electric fields that can be used to target these non-viral smooth muscle specific vectors to the airways. Using in vivo electroporation, we plan to test the efficacy of our smooth muscle-specific nuclear import sequence for gene transfer in animal models of asthma. We hypothesize that the SMGA DNA nuclear targeting sequence will lead to nuclear import and subsequent transcription of the DNA only in airway smooth muscle of living animals and not in other cells types of the lung. The specific aims of this proposal are to (1) determine the minimal SMGA DNA nuclear targeting sequence active in airway smooth muscle and the mechanisms of its cell specific DNA nuclear import, (2) test the efficiency of minimal SMGA DNA nuclear targeting sequences for driving airway smooth muscle-specific gene transfer in the lung, and (3) Test whether and how gene delivery of the 2-adrenergic receptor using smooth muscle-specific nuclear targeting constructs inhibits airway reactivity and smooth muscle proliferation in vivo.